Enhanced Glutamic Acid Production by a H<sup>+</sup>-ATPase-Defective Mutant of<i>Corynebacterium glutamicum</i>

Aoki, Ryo; Wada, Masaaki; Takesue, Nobuchika; Tanaka, Kenji; Yokota, Atsushi

doi:10.1271/bbb.69.1466

Cited by 42 publications

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References 27 publications

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“…The effectiveness of F 1 F o -ATP synthase defects for the production of acetate (9), as well as pyruvate (8), has also been reported for different E. coli mutants. Recently, we demonstrated that the mutation also works for the improvement of glutamate production in C. glutamicum (1).…”

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Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F₁-ATPase

et al. 2006

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The physiological changes in an F 1 -ATPase-defective mutant of Escherichia coli W1485 growing in a glucoselimited chemostat included a decreased growth yield (60%) and increased specific rates of both glucose consumption (168%) and respiration (171%). Flux analysis revealed that the mutant showed approximately twice as much flow in glycolysis but only an 18% increase in the tricarboxylic acid (TCA) cycle, owing to the excretion of acetate, where most of the increased glycolytic flux was directed. Genetic and biochemical analyses of the mutant revealed the downregulation of many TCA cycle enzymes, including citrate synthase, and the upregulation of the pyruvate dehydrogenase complex in both transcription and enzyme activities. These changes seemed to contribute to acetate excretion in the mutant. No transcriptional changes were observed in the glycolytic enzymes, despite the enhanced glycolysis. The most significant alterations were found in the respiratory-chain components. The total activity of NADH dehydrogenases (NDHs) and terminal oxidases increased about twofold in the mutant, which accounted for its higher respiration rate. These changes arose primarily from the increased (3.7-fold) enzyme activity of NDH-2 and an increased amount of cytochrome bd in the mutant. Transcriptional upregulation appeared to be involved in these phenomena. As NDH-2 cannot generate an electrochemical gradient of protons and as cytochrome bd is inferior to cytochrome bo 3 in this ability, the mutant was able to recycle NADH at a higher rate than the parent and avoid generating an excess proton-motive force. We discuss the physiological benefits of the alterations in the mutant.The elucidation of the regulatory mechanism of glycolytic flux is critical for the development of effective fermentation processes for the production of useful metabolites by microorganisms. Glycolytic flux in Escherichia coli is controlled primarily by the ATP demand of the cells rather than by glycolytic enzymes (22). For example, defects in the activity of F 1 F o -ATP synthase that impair oxidative phosphorylation (21,38,40) or increased ATPase activity in hydrolyzing ATP (22), which both lead to a reduced ATP/ADP ratio (21, 22), result in enhanced rates of glucose consumption. The enhancement of glucose consumption by defective F 1 F o -ATP synthase activity has also been reported for the gram-positive bacteria Bacillus subtilis (32) and Corynebacterium glutamicum (34), which are industrially important. Several attempts have been made to apply these findings to the production of useful metabolites from glucose by fermentation. Our group reported the first successful application of pyruvate production, using an E. coli mutant with a defective F 1 -ATPase (40). In this case, enhanced pyruvate production was achieved with an increased rate of glucose consumption. The effectiveness of F 1 F o -ATP synthase defects for the production of acetate (9), as well as pyruvate (8), has also been reported for different E. coli mutants. Recently, we demonstrated that...

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Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F₁-ATPase

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“…This improvement may have been due to increased glucose consumption by the H þ -ATPasedefective mutant, as described previously. 13,14) This is the first report to indicates that an H þ -ATPase-defective mutant of C. glutamicum is capable of producing an amino acid other than glutamate. The results also indicate that an H þ -ATPase-defective mutant is useful in the fermentative production of other compounds such as glutamate, 13) alanine, 19) and possibly other amino acids.…”

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“…Similar reduced cell densities, perhaps due to an energy shortage due to H þ -ATPase-defect, were also observed for the spontaneous H þ -ATPase-defective mutant F172-8. 13,14) Both the wild-type and A-1 strains of C. glutamicum without plasmid produced small amounts of valine (1.5-2.0 mM), but, no significant deference in valine productivity was observed between the wild-type and A-1.…”

Section: Valine Production Using C-terminal Truncated Ahas Plasmidmentioning

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“…In our previous study, we found that an H þ -ATPase defective mutant of C. glutamicum ATCC14067, strain F172-8, produced increased glutamate under conditions of exhaustive biotin deprivation. 13) However, because this mutant was selected by spontaneous neomycin resistance, 14) the possibility exists that other unknown mutations in addition to defective H þ -ATPase were also present. Hence, in this study we selected a defined H þ -ATPase defective mutant, C. glutamicum A-1, using the homologous recombination approach from C. glutamicum ATCC13032, whose entire genome has been sey To whom correspondence should be addressed.…”

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“…Increased intracellular pyruvate concentrations of H þ -ATPase-defective mutant strains over wild-type strains were also suspected in E. coli 19,20) and in C. glutamicum. 13,14) Because valine is formed from pyruvate without the requirement of ATP via the four-step biosynthetic pathway described above, valine appeared to be a particularly suitable target compound to investigate production in our defined H þ -ATPase-defective mutant strain in which decreased intracellular levels of ATP are probably present.…”

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Enhanced Valine Production inCorynebacterium glutamicumwith Defective H⁺-ATPase and C-Terminal Truncated Acetohydroxyacid Synthase

Wada

Hijikata

Aoki

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Enhancement of Central Energy Metabolism, Microbial

Yokota

Wada

2009

Encyclopedia of Industrial Biotechnology

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Improving the productivity of useful metabolites is critically important for industrializing the fermentation process. For this to be achieved, rapid conversion of the fermentation substrate into the target metabolite through enhancing central metabolism seems rational. Since the discovery of “Pasteur effects” more than 100 years ago, scientific evidence has accumulated, especially in the past 50 years, that central metabolism is under the control of the energy status of cells in vivo in such a way that catabolism is enhanced under energy‐deficient conditions. We investigated enhancement of fermentative production by boosting central metabolism through the manipulation of energy metabolism. We found that the introduction of defective F 1 Fo‐ATP synthase to impair oxidative phosphorylation is effective for enhancement of pyruvate production in Escherichia coli K‐12 (hereafter, we collectively define this mutation as the “ atp mutation” since F 1 Fo‐ATP synthase is encoded by the atp operon in E . coli K‐12). Clarification of the physiological alterations associated with the atp mutation revealed several interesting metabolic changes. Excellent producers of pyruvate and acetate have recently been engineered in E . coli K‐12, in which the atp mutation appears to contribute markedly to increase glucose consumption and metabolite production of the producers. Furthermore, the atp mutation was found to be effective for a gram‐positive industrially important bacterium, Corynebacterium glutamicum , in enhancing both glucose consumption and glutamic acid production. Elucidation of metabolic alterations in this atp mutant revealed significant differences from those found in the E . coli atp mutant, suggesting a diversity of adaptive responses to energy deficiency in different bacterial species.

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Enhanced Glutamic Acid Production by a H⁺-ATPase-Defective Mutant ofCorynebacterium glutamicum

Cited by 42 publications

References 27 publications

Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F₁-ATPase

Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F₁-ATPase

Enhanced Valine Production inCorynebacterium glutamicumwith Defective H⁺-ATPase and C-Terminal Truncated Acetohydroxyacid Synthase

Enhancement of Central Energy Metabolism, Microbial

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Enhanced Glutamic Acid Production by a H+-ATPase-Defective Mutant ofCorynebacterium glutamicum

Cited by 42 publications

References 27 publications

Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F1-ATPase

Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F1-ATPase

Enhanced Valine Production inCorynebacterium glutamicumwith Defective H+-ATPase and C-Terminal Truncated Acetohydroxyacid Synthase

Enhancement of Central Energy Metabolism, Microbial

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Enhanced Glutamic Acid Production by a H⁺-ATPase-Defective Mutant ofCorynebacterium glutamicum

Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F₁-ATPase

Alterations of Cellular Physiology inEscherichia coliin Response to Oxidative Phosphorylation Impaired by Defective F₁-ATPase

Enhanced Valine Production inCorynebacterium glutamicumwith Defective H⁺-ATPase and C-Terminal Truncated Acetohydroxyacid Synthase